U.S. patent number 4,839,419 [Application Number 07/161,204] was granted by the patent office on 1989-06-13 for method for immobilizing dissolved proteins.
This patent grant is currently assigned to Rohm GmbH. Invention is credited to Dieter Kraemer, Hermann Plainer, Reiner Schnee, Bruno Sproessier, Helmut Uhlig.
United States Patent |
4,839,419 |
Kraemer , et al. |
June 13, 1989 |
Method for immobilizing dissolved proteins
Abstract
Methods for adsorbing a protein, for example an enzyme, onto an
insoluble, solid, macroporous, small-particle support by washing
said support with an aqueous solution of the protein containing an
electrolyte in an ionic strength of at least 0.15 mole/liter and
crosslinking said protein, before, during, or after such
adsorption, with a coupling component present in aqueous
electrolyte-containing solution.
Inventors: |
Kraemer; Dieter (Mainz,
DE), Plainer; Hermann (Reinheim, DE),
Sproessier; Bruno (Rossdorf, DE), Uhlig; Helmut
(Rossdorf, DE), Schnee; Reiner (Darmstadt,
DE) |
Assignee: |
Rohm GmbH (Darmstadt,
DE)
|
Family
ID: |
6269271 |
Appl.
No.: |
07/161,204 |
Filed: |
February 16, 1988 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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853736 |
Apr 18, 1986 |
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Foreign Application Priority Data
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Apr 27, 1985 [DE] |
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3515252 |
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Current U.S.
Class: |
525/54.1;
210/656; 530/412; 530/413 |
Current CPC
Class: |
A23C
9/1206 (20130101); C12N 11/00 (20130101); C12P
13/04 (20130101); C12P 19/24 (20130101); B01J
20/3274 (20130101); B01J 20/3282 (20130101); A23C
2220/104 (20130101) |
Current International
Class: |
A23C
9/12 (20060101); C12P 13/00 (20060101); C12P
13/04 (20060101); C12P 19/00 (20060101); C12P
19/24 (20060101); C12N 11/00 (20060101); C07K
017/00 (); C12N 009/84 (); C12N 011/00 (); C12Q
001/00 () |
Field of
Search: |
;525/54.1 ;530/412,413
;210/656 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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26672 |
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Apr 1981 |
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EP |
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37667 |
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Oct 1981 |
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EP |
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3336257 |
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Apr 1984 |
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DE |
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1557944 |
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Dec 1979 |
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GB |
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1568328 |
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May 1980 |
|
GB |
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2128620 |
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May 1984 |
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GB |
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2129809 |
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May 1984 |
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GB |
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Primary Examiner: Kight; John
Assistant Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Curtis, Morris & Safford
Parent Case Text
This application is a continuation application of Ser. No. 853,736
filed Apr. 18, 1986 and now abandoned.
The present invention relates to a method for immobilizing
proteins, particularly enzymes, when dissolved in water, on a solid
substrate in the presence of an electrolyte.
Immobilized enzymes have already been obtained by crosslinking
dissolved enzymes with glutaraldehyde. It has been observed that
increasing ionic strength promotes this crosslinking, which has
been attributed to the precipitating action of electrolytes. (See
K. Ogata et al., Biochim. Biophys. Acta, 159 [1968], 405-407).
Glutaraldehyde has also been used to bind dissolved enzymes to an
insoluble support. (See A. Habeeb, Archives of Biochemistry and
Biophysics, 119 [1967], 264-268). In contrast with the
precipitation and crosslinking of proteins with glutaraldehyde
without a support, binding of a protein to solid supports has
always been carried out at low electrolyte concentrations.
In the process described in published German patent application DOS
33 36 257, permanently immobilized enzymes are produced by
impregnating a porous support, for example diatomaceous earth, with
an enzyme solution or by coating a support with a layer of the
solid enzyme, and then introducing these preparations comprising a
support and a dissolved or solid enzyme into an aqueous saline
solution and allowing a crosslinking agent to act on them. The
saline solution has the effect that no enzyme is stripped from the
enzyme preparation and lost for immobilization. This process thus
requires basically two separate process steps: production of an
enzyme preparation comprising a support and a dissolved or soluble
enzyme, and introduction of the enzyme preparation into a
crosslinking bath.
The binding of a fungal lactase with glutaraldehyde to a support
formed by macroporous ion exchange resins is described in European
Pat. No. 37667, while European Pat. No. 26672 describes such
binding to supports consisting of macroporous amphoteric ion
exchange resins. In both processes, electrolytes are used a buffer
substances only before or after the reaction with glutaraldehyde,
whereas during the reaction itself they are used only in low
concentrations which in no case exceed 0.05 mole/liter, in order
that the ionic adsorption not be vitiated. Indeed, to the contrary,
high ion concentrations are mentioned as a means for stripping from
the support such amounts of enzyme as may have remained
uncrosslinked. Amphoteric or cationic carrier resins may give rise
to a variety of troublesome interactions with dissolved
constituents of the substrate liquids and therefore are often
unsuitable for use.
The present invention accomplishes the object of immobilizing
dissolved proteins on macroporous supports with high recovery of
the initial activity (high activity yield) in a simple manner in a
one-step process.
It has been found that the immobilization of dissolved proteins on
a solid macroporous support which is water insoluble, or at most
slightly swellable in water, can be carried out in a one step
process if an aqueous protein solution containing an electrolyte
and having an ionic strength of at least 0.15 mole/liter is allowed
to wash over the support until the protein has been adsorbed
thereon. If purely adsorptive immobilization is sufficient, the
loaded support can simply be separated. As a rule, however,
immobilization is stabilized by means of a crosslinking agent. In
accordance with the invention, such crosslinking is carried out
directly in the electrolyte-containing solution without prior
separation of the protein-loaded support. Both during the
adsorption and in the binding by means of a crosslinking agent
which usually follows, a supernatant of the electrolyte-containing
solution is present so that the particles of the support which are
suspended or form a column packing are completely washed over.
It is surprising that immobilization of the protein on the
macroporous support according to the present invention is promoted
by high ionic strengths over the range here disclosed, since high
ionic strengths are generally regarded as a means for stripping
protein molecules that are not covalently crosslinked from a
support and for forming insoluble precipitates without a support in
the presence of crosslinking agents.
Following Table I shows the increase in recovery of activity in the
binding of penicillinamidase to phenylsepharose with glutaraldehyde
as a function of increasing ionic strength. The binding method
corresponded to the procedure used in Example 5.
Without the use of glutaraldehyde, no immobilized activity is
observed under otherwise identical conditions.
The advantages of the procedure preferred in accordance with the
invention over immobilization on an amphoteric support is
illustrated by the example of the immobilization of yeast lactase.
According to comparative Example 1 of European Pat. No. 26672,
yeast lactase was immobilized on an amphoteric phenol-formaldehyde
ion exchange resin at pH 6.65 and 20.degree. C.-22.degree. C. with
glutaraldehyde in a quantitative yield of 64 percent. However, the
activity was so low that practically no recovery of activity was
obtained.
However, if yeast lactase is bound at 23.degree. C. to beads of a
macroporous neutral carrier resin comprising crosslinked acrylamide
having oxirane groups as the active coupling groups from an aqueous
solution containing 111 g/l of the enzyme preparation, the
following activity yields are obtained as a function of the
electrolyte concentration used:
What is particularly surprising is that the method of the invention
makes it possible readily to obtain high absolute activities and
outstanding activity yield even on widely differing nonspecific
supports, which absolute activities and yields otherwise could be
obtained only after a complicated activation of the support. Once
crosslinked, the protein remains bound to the support even at low
ionic strength and, in the case of enzymes for example, can be
reused many times without appreciable losses of activity.
The process of the invention is particularly suited for the
production of all kinds of solid macroporous bodies which contain
soluble proteins in immobilized form. The principal use of the
invention is the production of carrier bound enzymes. Other
important uses are the production of column packings for affinity
chromatography and the production of diagnostic test bodies.
The proteins which can be immobilized according to the invention
are those proteins which are sufficiently water soluble at
temperatures between 0.degree. C. and 60.degree. C. to be brought
into contact in dissolved form with a support. The invention is not
limited to particular proteins of this kind but is applicable to
any biogenic, water soluble material containing protein. Although a
complete failure of the process of the invention has not been
observed so far, the possibility cannot be ruled out that it may
not be feasible to bind a particular protein to supports, or then
only with poorer results than by other methods. It can be
advantageous to stabilize the protein by adding ions or, in the
case of enzymes, by using substrate additives. Thus, it is known in
the art that many proteins are more stable to inactivation when in
the presence of certain ions, for example calcium or magnesium
ions. Salts of these ions can be added to protein solutions to
stabilize them. Similarly, many enzymes are more stable to
inactivation in the presence of the substrate toward which they are
active. For example, amyloglucosidase is stabilized by dextrin.
Enzymes are preferred and, of these, hydrolases, especially those
which cleave low molecular weight (less than about 2000)
substrates, particularly sugars such as lactose. Preferred
hydrolases are the lactases, invertases, acrylases, and penicillin
amidase. Other enzymes include amylases, proteases, amidases,
pectinases, cellulases, hemicellulases, isomerases, and oxidases.
Illustrative of nonenzymatic proteins are antibodies, hormones
having a protein structure, and enzyme inhibitors.
The support may be any solid, whether natural or synthetic, organic
or inorganic, having a macroporous structure and which is insoluble
in water or aqueous electrolyte solutions. Suitable solids with a
macroporous structure have pores of a diameter of at least 10
nanometers (nm) and a pore volume of over 0.1 cm.sup.3 /g, and
preferably over 0.5 cm.sup.3 /g. Preferred supports have pore
diameters ranging from 10 to 100 nm, a specific surface from 10 to
500 m.sup.2 /g, and a pore volume from 1 to 3 cm.sup.3 /g.
The outer shape of the support is not critical for its effect,
although high surface area systems having a specific surface of at
least 1 m.sup.2 /g are often preferred because they permit a large
amount of activity of the immobilized protein to be bound in a
small space. Suitable supports include the internal surfaces or
coatings of vessels or pipes or of built-in structures in vessels
or pipelines, and foils, membranes, papers, woven fabrics, nonwoven
fabrics, fibrous or other packings, or beddings of solid bodies
around or through which flow is possible. When there are
sufficiently large spaces between these bodies through which flow
is possible, the supports, or at least their layers close to the
surface, are able to swell considerably without impeding flow
through them. The supports are preferably of small size, meaning
supports having a particle size of less than 10 mm and preferably
ranging from 0.1 to 5 mm. Particularly preferred are beads, that
is, spherical supports. Advantageously, these small size supports
do not swell, or do not swell significantly, in the aqueous medium
in which they are used, preferably to not more than twice the bulk
volume of the dry material. A requirement for the suitability of
small size supports is that they lend themselves to being suspended
in the electrolyte containing solution by stirring, or permit flow
through them when used as column packing.
Most preferred are solid supports which are not cationically
charged. This is intended to mean solids which contain no
covalently bound cationic groups or basic groups which can be
converted by protonation into cationic groups, in other words
ammonium or amino groups, or in any case contain not more than 0.1
milliequivalent/gram of such groups in their matrix or at least on
their accessible surface. Particularly preferred are macroporous
solids which are uncharged or, if charged, carry only a weak
anionic charge. Uncharged solids here means solids which do not
contain any, or contain not more than 0.1 milliequivalent, of
covalently bound carbonyl, carboxylate, sulfonate, amino, or
ammonium groups per gram of dry carrier resin. Weakly anionic
carrier resins may contain anionic groups, such as covalently bound
carboxyl or carboxylate groups, in a concentration up to 5
milliequivalents per gram. These values apply in any case to the
resinous material on the active surface of the particles of the
support.
However, it has been found that superficial salting out of the
protein under the influence of the increased salt concentration can
occur also on very hydrophilic surfaces, and even on solids
carrying strong anionic charges. Such supports therefore are also
suitable for use.
Binding of the proteins is strongly promoted by hydrophobic groups,
for example by saturated or, preferably, unsaturated aliphatic
hydrocarbon side groups having two or more carbon atoms, or by
aromatic hydrocarbon groups. The size of these groups is less
important than their density on the surface of the support.
Preferred supports consist, at the surface of the pore wall or
throughout, of a polymeric material containing at least one weight
percent of side groups of the type mentioned.
In principle, macroporous inorganic supports such as glass beads or
glass fibers (for example, controlled pore glass), silicic acid,
alumina, or activated charcoal are also suitable for use. Organic
materials, and particularly synthetic resins, offer the advantage
that supports having a specific form, for example, beads, fibers,
foils, and coatings, can be more readily produced therefrom. The
chemical makeup of the organic support is not critical, although
care should be taken in each case to determine which support will
produce the best results.
Suitable water insoluble macroporous synthetic resins may be of a
nonpolar nature, for example, and may be uncrosslinked or
crosslinked, such as polymethyl methacrylate and other acrylate
polymers, polystyrene, cellulose esters, phenol-formaldehyde
resins, epoxy resins, or polyolefins. They may also be polar and
more or less hydrophilic but water insoluble due to crosslinking.
Examples are crosslinked cellulose derivatives and starch
derivatives, crosslinked polyacrylamide or polymethacrylamide,
crosslinked polyhydroxyalkyl esters of acrylic or methacrylic acid,
crosslinked polyvinylpyrrolidone, or nonbasic aminoplast
resins.
For improvement of the adsorptive properties it is often
advantageous to hydrophobize the support somewhat, at least
superficially. For example, phenyl groups may be superficially
incorporated onto inorganic supports such as silica, bentonite, or
porous glasses by reaction with phenyl-functional silanes.
Macroporous organic supports such as Sepharose can be analogously
phenylated with phenyl glycidyl ether or alkylated with alkyl
glycidyl ether. In addition to phenyl groups, all linear or
branched alkyl groups having from 1 to 18 carbon atoms generally
have an adsorption promoting action.
A particularly well suited group of carrier resins in the form of
beads or hollow beads is obtainable in the manner described in U.S.
Pat. Nos. 4,070,348, 4,190,713, and 4,208,309, incorporated herein
by reference. These are crosslinked polymers with a hydrophilic
matrix which have no, or only low, swelling capacity and which are
preferably composed in large measure of acrylamide, methacrylamide,
and methylenebis-acrylamide or -methacrylamide having crosslinking
action. They contain groups possessing binding activity for
proteins, and especially epoxy groups since the latter will not
form ionic groups by hydrolysis or during the reaction with the
protein. Moreover, they contain about 2 to 5 weight percent of free
methacryloyl or isopropanyl groups stemming from crosslinker
molecules that have been reacted only unilaterally. When such
supports are employed, the use of a separate crosslinking agent can
be dispensed with.
In the method of the invention, the electrolyte, it is speculated,
has the effect of bringing about a kind of supersaturation of the
protein, or at any rate a diminution of its solubility, because of
structural ordering of water molecules. It is often advantageous to
add the electrolyte only after the protein has dissolved as
otherwise the latter will occasionally dissolve only haltingly, if
at all.
Which electrolyte has a precipitating effect in what concentration
depends on the nature of the protein, its concentration, and its
impurities, and will have to be determined in each case by trial.
In general, all electrolytes which have a salting out effect are
usable, for example all salting out anions of the Hofmeister
series, including chloride. Examples for the varying effectiveness
of electrolytes are given by H. M. Rauen in "Biochemisches
Taschenbuch", part 2, 2nd ed., pp. 56-57. Electrolytes which have a
positive value of K.sub.s in accordance with the equation ##EQU1##
or with log S.sub.o =.beta.
log S=.beta.-K.sub.s .times.I,
where I is ionic strength, S is the solubility of the protein in
the presence of the electrolyte, S.sub.o the solubility of the
protein in the absence of the electrolyte, are suitable. Polyvalent
metal cations frequently having a precipitating or inactivating
effect, so that univalent cations, and particularly alkali ions and
ammonium ion, are therefore to be preferred. Anions have a more
pronounced influence on the effect of the electrolyte addition.
Sulfates, phosphates, and polyphosphates are best suited.
Carbonates, chromates, acetates, citrates and tartrates also exert
a strong salting out effect but cannot always be used with
sensitive proteins. Univalent anions such as chlorides or acetates
must be used in higher concentration than polyvalent anions to
reach the necessary ionic strength. To be able to exert their
salting out effect, polyvalent anions must be used in high
concentration. Because of their high effectiveness, low cost, and
innocuousness for most proteins, structure forming neutral salts
such as ammonium sulfate, sodium sulfate, and potassium sulfate and
ammonium, sodium, or potassium bisulfates are best suited. The pH
value of the protein solution containing electrolyte depends o the
sensitivity of the protein and is preferably in the range from 4 to
9. K.sub.s values, as defined above, follow the Hofmeister series,
proceeding from larger to smaller values: citrate, tartrate,
sulfate, acetate, chloride, nitrate, bromide, iodide, and
thiocyanate, among the anions and thorium, aluminum, barium,
strontium, calcium, potassium, sodium, and lithium, among the
cations. (cf. Rauen, loc. cit.)
The effectiveness of the electrolyte addition is determined by its
ionic strength I. For an aqueous solution containing i ionic
species, ionic strength is calculated by the formula ##EQU2## where
C.sub.i is the molar concentration and Z.sub.i is the valence of an
ionic species. For example, for a 1 M K.sub.2 HPO.sub.4 solution
wherein C.sub.K =2, C.sub.HPO.sub.4 =1, Z.sub.K =1, and
Z.sub.HPO.sub.4 =2, the ionic strength, I, is 3. In practice, the
concentrations of the preferred electrolytes are in the ionic
strength range from 0.15 to 2 moles/liter. The ionic strength is
preferably at least 0.3 mole/liter.
Immobilization generally begins with an adsorption step. Even when
a crosslinking agent is used, the crosslinking reaction is preceded
by the adsorption step. The two steps may be carried out
successively in that sequence, that is first the protein is
adsorbed onto the support in the presence of the electrolyte and
then the crosslinking coupling component is added. This is the
preferred sequence. However, the two steps can also be combined in
the process and the coupling component can already be allowed to
react along with the electrolyte during the adsorption. Finally, in
many cases it is also possible to first react the support with the
coupling component alone and then to add the protein and the
electrolyte.
Adsorption of the protein occurs in the system formed by a
supernatant of the aqueous protein solution and by the support
material over a period from 0.1 to 100 hours, and preferably from 1
to 10 hours. It is important that the electrolyte wash all over the
particles of the support in both reaction stages. Immobilization is
accelerated as the temperature rises. The operating temperature is
preferably the highest temperature with the protein can withstand
without loss of activity. Although immobilization can be carried
out even at 0.degree. C., temperatures above 40.degree. C., and in
many cases above 50.degree. C., are particularly advantageous.
The proteins to be immobilized can be used in a wide range of
concentrations, for example from 0.01 to 30 percent, by weight of
the solution, and in a wide range of ratios of their amount with
respect to the amount of support material. In the case of enzymes,
a high binding yield and a high product activity are sought. The
loading generally is greater than 20 mg of protein per gram of dry
support material, that is, 2 weight percent or more. On the other
hand, low loading densities are more advantageous for immobilized
ligands used for the isolation of biomacromolecules.
Immobilization may be carried out by suspending the support in the
protein solution and then stirring moderately from the time after
the electrolyte is added until the process is completed. The
protein solution containing electrolyte may also be caused to flow
through a packing, formed by the support material, in a column
reactor and recirculating the solution for an extended period of
time.
Immobilization initially involves only the adsorption of the
protein onto the support. When the loaded support is then
transferred to an environment poorer in electrolyte, desorption is
likely to occur in many cases. This is prevented by the subsequent
covalent binding of the protein molecules to one another or to the
support. For this purpose, a coupling or crosslinking component is
added during the second reaction stage. As mentioned earlier, this
can be done even during the adsorption step.
The coupling component must be soluble in the aqueous electrolyte
solution in an amount that is at least sufficient for
immobilization of the protein. It is important that this component
be used in a supernatant of the electrolyte solution, in other
words the solution must not be completely absorbed by the support
material. Not less than about 0.2 ml, and preferably up to 10 ml,
of electrolyte solution should be used per milliliter of moist
support material. Moreover, the coupling component must react in
the presence of the electrolyte in order that the precipitating
action of the latter may be sustained until the protein has been
irreversibly immobilized.
Suitable coupling components are compounds having at least limited
water solubility and two or more functional groups with which they
are able to react with corresponding functional groups of the
protein, and optionally also with functional group of the support.
Irreversible immobilization may be brought about by a crosslinking
of the protein within itself or by crosslinking it with the
support. Suitable functional groups which are reactive toward
proteins are aldehyde, epoxy, diazo, isocyanate, and chloroformate
groups, for example. Carboxyl anhydride or ester groups are in many
cases less advantageous because they strongly alter the
electrochemical nature of the support by the formation of anionic
carboxylate groups. Illustrative of suitable coupling components
are diazobenzidine, hexamethylene diisocyanate, chloroformic acid
ethyl ester, and glutaraldehyde, the most important compound. Water
soluble low molecular weight polymers having molecular weights
below 100,000, for example polyacrolein and copolymers of
acrylamide or methacrylamide with glycidyl acrylate or methacrylate
or with acrolein or with N-allylmethacrylamide may also be
used.
Mention should also be made of coupling components which are
activated only by the action of ultraviolet radiation or by agents
forming free radicals (redox initiators), for example diallyl
ethers or copolymers of acrylamide or methacrylamide with
para-toluylhydroxyethyl methacrylate.
The amount of coupling component required for irreversible
immobilization depends on its reactivity with respect to the
protein, and possibly with respect to the support, and may range
from 1 to 100 percent of weight of the protein. The reaction
conditions for coupling generally do not differ from those of the
adsorption so that both processes can take place under the same
conditions. In coupling with glutaraldehyde, a reaction time from
0.1 to 100 hours, and preferably of about 2 hours, at temperatures
ranging from 0.degree. C. to 80.degree. C. is usually
sufficient.
After the immobilization reaction, the electrolyte solution, which
may contain unbound residues of the protein and of the coupling
component, is usually separated from the loaded support. The latter
is then washed with an appropriate buffer solution and is then
available for commercial use. Activity values not found in the
residual electrolyte solution or found to the support are lost
(i.e. inactivated during the process.
A better understanding of the invention and of its many advantages
will be had by referring to the following Examples, given by way of
illustration.
Claims
What is claimed is:
1. A method for immobilizing a dissolved protein on a solid,
macroporous, water insoluble support having pores of a diameter of
at least 10 nanometers and a pore volume greater than 0.1 cm.sup.3
/g, in the presence of an electrolyte innocuous for said protein,
which method comprises washing a supernatant aqueous solution of
the protein over said support until the protein has been adsorbed
onto the support, said solution having an ionic strength of the
electrolyte of at least 0.15 mole/liter but insufficient to
coagulate the protein from the solution, and adding to the
electrolyte-containing solution, after adsorption of the protein
onto the support, a water soluble coupling component effecting
crosslinking of the protein adsorbed onto the support.
2. A method as in claim 1 wherein said support either has no
covalently bound cationic groups or no more than 0.1
milliequivalent/gram of such groups.
3. A method as in claim 2 wherein said support has not more than 5
milliequivalent/gram of covalently bound anionic groups per gram of
dry weight.
4. A method as in claim 3 wherein said support has not more than
0.1 milliequivalent of covalently bound ionic groups per gram of
dry weight.
5. A method as in claim 1 wherein said support does not swell to
more than twice its volume in an aqueous medium.
6. A method as in claim 5 wherein said support is in the form of
beads.
7. A method as in claim 5 wherein said support is in the form of
particles having a particle size of less than 10 mm.
Description
EXAMPLE 1
Immobilized glucose isomerase
Support: Macroporous highly crosslinked styrene/divinylbenzene
beads having an inner surface area of about 200 m.sup.2 /g and an
average pore diameter of 40 nm.
Enzyme: Glucose isomerase, liquid concentrate from a culture of
Streptomyces albus.
Activity: 1 g of enzyme converts D-glucose from a 0.1 molar
solution at pH 7 and 70.degree. C. to 5 g of D-fructose in 60
minutes.
Coupling: First the enzyme is adsorbed onto the macroporous
support. To this end, 10 g of support and 10 g of enzyme in 50 ml
of saline solution containing 12% of sodium sulfate, 5% of
magnesium sulfate, and 0.02% of cobalt sulfate are rolled at room
temperature (23.degree. C.) on a roller table. The ionic strength
of the aqueous solution is 4.18 moles/liter. The pH value was
adjusted to 7.0. After 20 hours, 0.5 g of glutaraldehyde is added
and rolling is continued. The adsorbed enzyme is crosslinked and
retained in the pores by this process.
Filtration by suction and washing follow 2 hours later. A
comparative activity determination shows an activity of 45% in the
filtrate and of 37% in the support.
Activity Yield: 37%.
Use: The immobilized glucose isomerase is used as packing in a
column reactor. At a temperature of 60.degree. C. and a throughput
rate of seven times the fixed-bed volume per hour, a 40% glucose
solution of pH 7.5 is isomerized to fructose with 45%
conversion.
EXAMPLE 2
Immobilized Aspergillus oryzae lactase
Support: Crosslinked polyacrylic ester; average pore diameter, 25
nm; inner surface area, 140 m.sup.2 /g; beads from 0.3 to 1 mm in
diameter.
Enzyme: Aspergillus oryzae lactase, powdered concentrate. Activity:
30,000 U/g.
Coupling: 10 g of support are shaken with 1 g of enzyme preparation
in 40 ml of saline solution at 35.degree. C. for 8 hours. The
saline solution contains 24% of potassium chloride and was adjusted
to pH 5.0. The ionic strength of the aqueous solution is 3.21
moles/liter. 0.1% of lactose was further added for stabilization of
the enzyme. After cooling, shaking is continued for another 2 hours
at room temperature with addition of 0.5% of glutaraldehyde. This
is followed by filtration by suction and washing. The lactase
activities of support and filtrate are then compared. The support
is found to have 34% of the initial activity, the filtrate,
11%.
Activity yield: 34%.
Use: The immobilized Aspergillus lactase is used as packing in a
column reactor. At a temperature of 35.degree. C., a lactose
solution of pH 4.5 is hydrolyzed at a throughput rate of 40
fixed-bed volumes per hour with over 90% conversion. After 60 days,
no loss of activity is observable.
EXAMPLE 3
Immobilized yeast lactase
Support: Macroporous, highly crosslinked bead polymer comprising
methacrylamide/methylenebismethacrylamide having free epoxy groups
(1.2% oxirane oxygen) and 2.2% of adhering isopropenyl groups which
are concentrated on the inner surfaces of the pores. Pore volume,
3.4 ml/g. Average pore diameter, 20 nm. The preparation of this
support is described in Example 2 of German patent publication 27
22 751.
Coupling of the adsorbed enzyme here is effected covalently by
reaction with the epoxy groups simultaneously with adsorption of
the enzyme under the influence of the high salt concentration.
Enzyme: Yeast lactase from Saccharomyces (Kluyveromyces) lactis,
liquid preparation 5,000 neutral lactase units (NLU).
Coupling: 10 g of support are shaken at room temperature
(23.degree. C.) with 10 g of enzyme in 80 g of saline solution. The
latter contains 16% of dibasic potassium phosphate, 7.9% of
monobasic potassium phosphate and, for stabilization of the enzyme,
20 ppm of MnCl.sub.2. 4H.sub.2 O. The solution has an ionic
strength of 3.34 moles/liter. After 72 hours, filtration by suction
and washing are carried out and the lactase activity of the
filtrate is compared with that of the support. The support is found
to have 55% of the initial activity and the filtrate 14%.
At 55%, the activity yield is very high in the case of this
sensitive enzyme.
Use: The coupled yeast lactase is used as packing in a column
reactor. At a temperature of 7.degree. C., skim milk with 0.3% fat
is passed through it for 20 days at a flow rate of 55 fixed-bed
volumes per hour. The lactose contained in the milk is hydrolyzed
to glucose and galactose, at first with 65% conversion, and after
20 days with 50% conversion.
EXAMPLE 4
Immobilized aminoacylase
Support: Macroporous highly crosslinked bead polymer comprising
methacrylamide/methylenebismethacrylamide having free epoxy groups
(1.2% oxirane oxygen) and 2.2% adhering isopropenyl groups which
are concentrated on the inner surfaces of the pores. Pore volume,
3.4 ml/g. Average pore diameter, 20 nm. The preparating of this
support is described in Example 2 of U.S. Pat. No. 4,208,309.
Enzyme: Powdered aminoacylase concentrate from an Aspergillus
strain.
Activity: 23,000 U/g. Substrate: Acetyl-D, L-methionine.
Coupling: 10 g of support are shaken at 35.degree. C. with 20 g of
enzyme preparation in 80 ml of saline solution. The latter contains
14.2% of sodium sulfate and 24 ppm of CoCl.sub.2.6H.sub.2 O and is
adjusted to pH 7.0. Its ionic strength is 3 moles/liter,
disregarding the salt content of the enzyme preparation.
After 8 hours, the support is filtered by suction and washed. The
support is found to have 61% of the initial activity and the
filtrate, 1%. The activity yield thus is 61%.
EXAMPLE 5
Binding of penicillinamidase to various supports
3.5 g portions of moist support material according to Table III are
washed five times, each time with five times their volume of
desalinated water, and then suction filtered on a porous glass
plate. Then the support material is shaken at about 21.degree. C.
for 2 hours with 6.8 ml of an enzyme solution containing 676
international units (IU) of penicillinamidase from E. coli in 0.5 M
of potassium phosphate buffer (pH 7.5, with 0.1% NaN.sub.3). The
ionic strength of the buffer solution is 1.5 moles/liter. After the
addition of 0.136 ml of a 25% aqueous glutaraldehyde solution which
had been stabilized with an ion exchange resin (Amberlite A 21),
shaking is continued for 2 hours. The loaded support material is
then placed on a porous glass filter and washed three times with 1
M NaOH and twice with 0.05 M sodium phosphate buffer (pH 7.5, with
0.1% NaN.sub.3).
The enzymatic activity was determined by alkalimetric titration at
pH 7.5 using penicillin G K (crude) as a substrate. For this
purpose, 20 ml each of the 2% substrate solution in 0.05 M sodium
phosphate solution at pH 7.5 were used and automatically titrated
at 37.degree. C. with 0.5 M sodium hydroxide solution. The results
are presented in Table III.
TABLE III
__________________________________________________________________________
Activity of Immobilized penicillinamidase Support Material U/g
Activity Chemical composition Trade name moist weight Yield
__________________________________________________________________________
(a) Crosslinked agarose ("Sepharose-CL-4B") 96 58 (b) Octylated
cross- ("Octyl-Sepharose- 98 53 linked agarose CL-4B") (c)
Phenylated cross- ("Phenyl-Sepharose 168 91 linked agarose CL-4B")
(d) Cross linked agarose ("DEAE-Sepharose- 84 61 substituted with
CL-6B") diethylaminoethyl groups (cataionic) (e) Carboxymethylated
("CM-Sepharose- 94 48 crosslinked agarose CL-6B") (anionic) (f)
Phenoxyacetylcellulose 77 59 (g) Oxirane-polyacrylamide resin 172
85 (according to U.S. 4,208,309, (Example 1), reacted with benzyl
thiol (h) Polymethacrylimide ("Rohacell WF") 70 45 foam, ground (i)
Crosslinked polymethyl methacrylate/ 35 25 glycol dimethacrylate
copolymer**, weight ratio 90:10 (j) Crosslinked polymethyl
methacrylate/ 55 37 glycol dimethacrylate copolymer**, weight ratio
80:20 (k) Crosslinked polymethyl methacrylate/ 43 33 glycol
dimethacrylate copolymer**, weight ratio 60:40 (l) Crosslinked
polystyrene 57 42 with 10% divinylbenzene (m) Porous glass,
("Controlled Pore Glass 10") 105 60 pore volume 0.75 cm.sup.3 /g,
average pore diameter 17 nm, inner surface area 107 m.sup.2 /g
__________________________________________________________________________
**Method of preparation: A solution of 1 part by weight of
polyvinyl alcohol in 320 parts of water is heated to 50.degree. C.
in a stirred vessel and a mixture of 100 parts of monomers
(methylmethacrylate and glycol dimethacrylate or styrene and
divinylbenzene, respectively), 60 parts of nheptane, and 1.4 parts
of dibenzoyl peroxide is dispersed therein as droplets with
stirring. During the 4hour polymerization time, the temperature is
held by cooling to a maximum of 75.degree. C. After that, the
solvent is distilled off over 1 hour at 36.degree. C. The polymer
beads formed are separated by filtration after cooling.
EXAMPLE 6
Binding of trypsin to "Phenyl-Sepharose"
3.5 g (moist weight) of "Phenyl-Sepharose" are pretreated as in
Example 1. Then 150 mg of trypsin dissolved in 6.8 ml of 0.5M
potassium phosphate buffer (pH 7.5, with 0.1% NaN.sub.3) are added,
followed by shaking at 23.degree. C. for 2 hours. The ionic
strength is 1.5 moles/liter. Immobilization of the adsorbed enzyme
and further treatment are carried out as in Example 1.
The enzymatic activity was determined using casein and
N-benzoyl-1-arginine ethyl ester hydrochloride (BAEE) as
substrates.
______________________________________ Results: Using casein 8.3
U/g moist weight Using BAEE 311 U/g moist weight
______________________________________
EXAMPLE 7
Production of support-bound lactase preparations
1 g portions of Aspergillus oryzae lactase "Lactase Preparation
2214 C Conc." having a strength of 30,000 U/g are dissolved in 40
ml portions of a 0.7 M Na.sub.2 SO.sub.4 solution having an ionic
strength of 2.1 moles/liter at pH 5.5. Portions of such solutions
are then mixed in each case with 10 g of one of the carrier resins
listed in Table IV and shaken for 20 hours at room temperature. 0.8
ml of 25% aqueous glutaraldehyde solution is then added and shaking
is continued for 2 hours at room temperature. The preparations are
separated by filtration and washed and the activity of the
immobilized enzyme and of the enzyme found in the filtrate is
determined and expressed in percent of the initial activity. The
results are presented in Table IV.
TABLE IV
__________________________________________________________________________
Activity Immobilized Residual lactase activity Activity in yield
filtrate Carrier Material (%) (%)
__________________________________________________________________________
(a) Weakly basic ion 17.5 0 exchanger based on styrene/divinyl-
benzene ("Amberlite IRA 93") (b) Macroporous adsorber 29.5 18 resin
comprising poly- acrylate basis ("Amberlite XAD 7") (c) Macroporous
phenol- 24.0 5 formaldehyde adsorber resin, weakly basic ("Duolite
S 561") (d) Macroporous phenol- 26.0 4 formaldehyde adsorber resin,
weakly basic ("Duolite S 587") (e) Macroporous phenol- 22.0 6
formaldehyde adsorber resin, nonionic ("Duolite S 761") (f)
Macroporous glass, 42.4 8 pore volume 0.75 cm.sup.3 /g, average
pore diameter 17 nm, inner surface area 107 m.sup.2 /g ("Controlled
Pore Glass CPG 10") (g) Polymethacrylimide 31.0 0 foam, ground
("Rohacell") (h) Hydroxyl apatite 16.5 4 (basic calcium phosphate),
ground
__________________________________________________________________________
EXAMPLE 8
Production of support-bound lactase preparation:Crosslinker is
added at the beginning of adsorption
1 g of lactase from Aspergillus oryzae ("Lactase Preparation 2214 C
Conc.") having a strength of 30,000 U/g is dissolved in 40 ml of a
0.7 m Na.sub.2 SO.sub.4 solution having a ionic strength of 2.1
moles/liter al pH 5.5. This solution is then mixed with 10 g of the
carrier (b) out of Table IV. At the same time 0.8 ml of 25% aqueous
glutaraldehyde solution is added and this suspension is shaken for
20 hours at room temperature.
Crosslinking happens here during adsorption.
The preparation ia separated by filtration and washed and the
activity of the immobilized enzyme and of the enzyme found in the
filtrate is determined as 25% respectively 4%.
EXAMPLE 9
Production of support-bound lactase preparation:Crosslinker is
added before adsorption
Example 8 is repeated, but the addition of the aqueous
glutaraldehyde to the enzyme solution occurs 20 minutes before the
carrier is added.
Crosslinking happens here before adsorption.
The activity of the immobilized enzyme and of the enzyme found in
the filtrate is determined as 17%, respectively 0%.
* * * * *